Dr. Richard Kerr - Development of an Effective Pathogen Screening Program using Rapid, High-Throughput Molecular Detection Assays
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Development of an Effective Pathogen Screening Program using Rapid, High-Throughput Molecular Detection Assays - Dr. Richard Kerr, Diagnostic R & D Lab Director, Daisy Farms, from the 2016 NIAA Annual Conference: From Farm to Table - Food System Biosecurity for Animal Agriculture, April 4-7, 2016, Kansas City, MO, USA. More presentations at http://www.trufflemedia.com/agmedia/conference/2016_niaa_farm_table_food_system_biosecurity
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Dr. Richard Kerr - Development of an Effective Pathogen Screening Program using Rapid, High-Throughput Molecular Detection Assays
- 1. Development of a Pathogen Screening Program using Rapid, High-Throughput Molecular Detection Richard Kerr, PhD, CBL, CMP Daisy Farms Paris, TX
- 2. Overview • About Daisy • In the fall of 2014… • Molecular Testing; real-time q-PCR • Automating the Workflow • Testing Programs • What we found • Next Steps
- 7. Bacterial ID Culture Results Freq. Notes; Staphylococcus sciuri 15 Animal Normal flora; Most S. sciuri isolates collected from immunocompromised patients Aerococcus viridans 15 Typically isolated from immunocompromised patients Proteus mirabilis 9 Frequently isolated from patients with underlying diseases or compromised immune systems Kocuria kristinae 9 opportunistic pathogen for the immunocompromised
- 12. qPCR • Agilent Mx3005P Real-time quantitative PCR thermocycler Respiratory Real-time qPCR Result Bovine Syncytial Respiratory Virus (BSRV) 1 Positive Infectious Bovine Rhinotracheitis (IBR) Negative Parainfluenza 3 (PI3) Negative
- 13. What do we do now? Culture Results Freq. Notes; Staphylococcus sciuri 15 Animal Normal flora; Most S. sciuri isolates collected from immunocompromised patients Aerococcus viridans 15 Typically isolated from immunocompromised patients Proteus mirabilis 9 Frequently isolated from patients with underlying diseases or compromised immune systems Kocuria kristinae 9 opportunistic pathogen for the immunocompromised Respiratory Real-time qPCR Result Bovine Syncytial Respiratory Virus (BSRV) 1 Positive Infectious Bovine Rhinotracheitis (IBR) Negative Parainfluenza 3 (PI3) Negative
- 14. Serology Serology Instance Leptospira, serovar hardjo 0.4%, n=245 Bluetongue virus (BTV) 5.7%, n=318 Anaplasma spp. 1.4%, n=350 Bovine Leukemia Virus (BLV) 2.1%, n=329 Bovine Viral Diarrhea (BVDv) 1.0%, n=308
- 15. Why RT-PCR? • Good • Specific; Detects presence of RNA sequence specific to BVDv • Sensitive; Can detect down to 1-10 viral copies • Fast; 3-5 hours from sample to result • Not-so-Good • Low Throughput; 44-48 tests/8 hr shift
- 17. Testing Program • Test for the following; • Leptospira borgpetersenii, serovar hardjo • Blue Tongue Virus (BTV) • Anaplasma spp. • Bovine Leukemia Virus (BLV) • Bovine Viral Diarrhea virus (BVDv) • Test purchase animals prior to shipping and quarantine on arrival • Test purchase animals on arrival; release on double negative results • Test 10% or 10 animals on movement across farm facilities • Test all animals 21 days prior to expected calving date
- 18. Data
- 19. What we found • Workflow Automation • Improve throughput up to 768 tests/8 hr shift • In 2014/2015, we received 1,417 samples for BVD testing. • 3 animals were positive for BVD in November, 2014. • No BVD positive result since. • Unfortunately, the scours continued… • RNA sequencing found Bovine Coronavirus. • Began vaccinating for Coronavirus about 7 weeks ago and scours cases are now down more than 80%.
- 20. Next Steps… • Part of a larger disease prevention/ health promotion strategy. • Current serology & PCR testing covers last two thirds of critical exposure period during gestation, so consider testing calves also?
- 21. Special Thanks to the Lab’s team
- 22. Questions?
Editor's Notes
- 7,600 acre Commercial Size Dairy Research Facility in NE Texas High genomic herd & calf raising facility Produce milk for Daisy Brand Sour Cream & Cottage Cheese Commenced Operations in 2012
- In late 2014, we noticed increased instances of scours, pneumonia, and early embryonic death Histopathology indicated severe chronic bacterial pneumonia Initial cultures found Trueperella pyogenes Cases accumulated with similar findings; severe bacterial pneumonia often with opportunistic and/or environmental bacteria.
- At scale, less labor to perform extraction Higher throughput; 384-768 samples per day (with another Thermocycler).
- BVDv is associated with immunosuppression The longer a PI animal is in the herd, the more animals it can infect, so speed matters.